1b, top). Generally, for AdV construction using the COS-TPC method, we isolated a single virus clone to avoid contamination of the parent Ad5 derived from the DNA-TPC or unexpected reassortants (27). Clones lacking the upstream loxP were unexpectedly obtained when using both pAxLEFZ15L and pAxLEFZ19L. This virus, named AxLEFZ (ΔL) (Fig. 1b, bottom right), was found to be identical to 15L and 19L, except for the deletion of the upstream loxP as determined using restriction analyses and sequencing. We considered that ΔL was generated by homologous recombination within the packaging domain (Fig. 1b). Thus, we used ΔL as a control virus in this work. However, this recombination appears to be

a rare event Alectinib because, once the virus genome obtains the terminal protein at the right end through the recombination of the large homology, the virus repairs its left terminal by adding a new terminal protein at the right end through a “pan-handle” structure (27, 29). The set of three LacZ-expressing AdV, 15L (AxLEFZ15L), 19L (AxLEFZ19L), and ΔL (AxLEFZ), (Fig. 2a, top left), is called the “LEFZ series” in this paper. For the competitor virus, we constructed AxCAGFP (Fig. 2a, top left), which expressed enhanced

green fluorescent protein (Takara Bio, Shiga, Japan) under the control of the CAG promoter, using the COS-TPC method. The AdV titer was calculated using the TCID50 using 293 cells (30). Briefly, 50μL of DMEM supplemented with 5% FCS were dispensed into each well of a 96-well plate, and eight rows of threefold serial dilution Y-27632 ic50 of the virus. Then, 3 × 105 of 293 cells was added to each well. The plate was incubated at 37°C and 50 μL of DMEM supplemented with 10% FCS was added to each well every 3 days. Twelve days later, the end-point if the cytopathic effect was determined by microscopy. The 293 cells were infected with 15L, 19L or ΔL at an MOI of 3 and with the competitor

virus at an MOI of 1 or 0.1 for 1 hr and then were cultured in a six-well plate. Three days after infection, the 293 cells were Montelukast Sodium harvested together with the medium. The cell suspension was sonicated for 2 min (30 s × 4 cycles) using a Bioruptor II sonicator (CosmoBio, Tokyo, Japan) at maximum power (200 W) and centrifuged at 1900 g using a Tomy TMP11 microcentrifuge rotor (Tomy, Tokyo, Japan) for 5 min at 4˚C. The supernatant was stored as the first viral stock. An aliquot (100 μL each) was used to infect 293 cells on a six-well plate, and the culture medium was obtained as the second viral stock. Similar virus passages were continued six times to obtain the seventh viral stock. To monitor the genome structure of the virus, the infected cells at each passage were centrifuged at 1900 g for 5 min at 4°C, and the total cell DNA together with the viral genome DNA was prepared according to the method of Saito et al. (31).

Endoplasmic HDAC activity assay reticulum (ER) stress has been postulated as one contributor during the development of renal fibrosis. The present study investigated the anti-fibrotic

effects through the attenuation of ER stress, exerted by sodium 4-phenylbutyrate (4-PBA), a chemical chaperon of ER, and mechanisms of underlying these effects. Methods: Anti-fibrotic effects in vivo were assayed in a rat model of renal fibrosis [the unilateral ureteral obstruction (UUO) model]. A rat tubular epithelial cell line (NRK-52E) was stimulated by transforming growth factor-β1 (TGF-β1) and treated with 4-PBA to explore possible mechanisms of these anti-fibrotic effects. Protein expression was analyzed by Western blotting. Transcriptional regulation was investigated using luciferase activity driven by a connective tissue growth factor (CTGF) promoter. Results: The 4-PBAsignificantly

attenuated UUO-induced overwhelming ER stress-related protein expressions, and restored adaptive ER response, splicing X-box-binding protein 1 expression. 4-PBA also attenuated apoptosis, renal fibrosis and tubulointerstitial injury, which is accompanied by attenuating α-smooth muscle actin and CTGF protein expressions ADP ribosylation factor in the rat UUO kidney. 4-PBA also inhibited TGF-β-induced ER stress-associated proapoptotic molecules, profibrotic H 89 molecular weight factors, and CTGF-luciferase activities in renal tubular cells. Conclusion: 4-PBA, acts as an ER chaperone, amelorites ER stess and protects against renal tubular cell apoptosis and renal fibrosis. 4-PBA may become a therapeutic agent to prevent renal fibrosis. TAGUCHI ATSUHIRO, NISHINAKAMURA RYUICHI Department of Kidney Development, Institute of Molecular Embryology and Genetics,

Kumamoto University Introduction: Generation of the kidney in vitro is a challenge for developmental biology and regenerative medicine, because reconstitution of the three-dimensional structures including glomeruli and nephric tubules is a prerequisite for the kidney functions. Adult kidney derives from embryonic metanephros which develops by the reciprocal interaction of the metanephric mesenchyme (MM) and the ureteric bud (UB). Most kidney components are derived from metanephric nephron progenitors in the MM. However, the developmental process how the MM is formed in vivo is largely unknown, resulting in the unsuccessful reconstitution of kidney from pluripotent stem cells (PSCs) in vitro.

Spontaneous contractions and possible consequent afferent nerve firing might participate in the generation of OAB. The authors declare no conflict of interest. “
“Objectives: We report on our initial data from a prospective study to determine the efficacy of high-frequency magnetic stimulation on the sacral root (MSSR) for the intractable post-radical prostatectomy, stress urinary

incontinence (SUI). Methods: A total of 14 men with persistent SUI after a radical prostatectomy underwent treatment once every 2 weeks over a 40-week period for a total of 20 sessions. The outcome was assessed by these variables at baseline, at immediately after the first session, and at immediately after the final (20th) session. Results: Mean leak episodes (per day) consistently decreased after the first to the final session Tyrosine Kinase Inhibitor Library datasheet (from 6.1 Selleck Smoothened Agonist ± 2.9 to 3.5 ± 2.6, and to 3.0 ± 2.3, P < 0.01), and it remained to be decreased following 2 months after

the final session. The mean pad weight (per h) also decreased after the treatment (but no statistically significant change compared to the pretreatment level). The cystometric bladder capacity at the first desire to void and the capacity at the strong desire to void increased significantly following the high-frequency MSSR (first desire to void: from 146 ± 43 to 182 ± 52 mL; strong desire to void: from 224 ± 69 to 258 ± 60 mL, P < 0.01). No obvious complication was observed in any patients during or after the treatment. Conclusion: This study provides the preliminary evidence that high-frequency MSSR may potentially afford a useful option with minimal invasiveness Methane monooxygenase for the patients with obstinate SUI after a radical prostatectomy. “
“Objectives: The aim of the present study was to determine the causes for overactive bladder (OAB) symptoms in women visiting a urological clinic. Methods: We prospectively recruited female patients with OAB symptoms between December 2008 and February 2010. All patients were interviewed for their

detailed personal and medical history. All patients completed a 3-day frequency-volume chart. Symptom severity was evaluated using the International Prostate Symptom Score (IPSS) and Overactive Bladder Symptom Score (OABSS) questionnaires. All patients underwent either conventional pressure-flow urodynamic studies or video-urodynamic studies. On the basis of these evaluations, patients were assigned to one of the following categories: idiopathic OAB, stress urinary incontinence (SUI)-associated, neurogenic bladder, or bladder outlet obstruction (BOO). Results: A total of 108 female patients were recruited into the study. The mean age of the patients was 63.75 ± 14.02 years (range: 23–89). Detrusor overactivity was demonstrated in 55 patients (51%). The differential diagnosis was idiopathic OAB in 51 women (47.2%), SUI-associated in 46 (42.6%), neurogenic bladder in 13 (12.0%) and BOO in 7 (6.5%).

For NHD, dialysate concentrations need to be

adjusted especially increasing calcium and decreasing bicarbonate concentration, phosphate supplementation may be required and blood and dialysate flow rates can often be lowered. Treatment frequency and/or duration of HD regimens may also need to be adjusted to meet clearance targets and normalize blood pressure, extracellular fluid volume and serum parameters. The author gratefully acknowledges the expertise of Professor Peter Kerr (Monash Medical Centre, Clayton), Associate Professor John Agar (Barwon Health, Geelong) and the home haemodialysis nursing staff at Barwon Health (Geelong, Victoria) for their assistance in reviewing this manuscript. “
“The Australian and New Zealand Society Torin 1 cost of Nephrology gratefully acknowledges the support of the following companies: Sustaining Member/Education Partner/Research Partner Roche Products Pty Ltd Sustaining Members/Education Partners Baxter Healthcare Pty Ltd Janssen-Cilag Pty Ltd Novartis Pharmaceuticals Australia Pty Ltd Pfizer Australia Pty Ltd Sanofi Shire Australia Pty Ltd Sustaining Member/Research Partner Amgen Australia Pty Ltd Sustaining Members check details Fresenius Medical Care Australia Pty Ltd Gambro Pty Ltd Servier Laboratories Australia

Pty Ltd “
“Aim:  To determine the proportion of patients achieving tacrolimus whole-blood concentrations of ≥10 ng/mL within 3 days of kidney transplantation, after randomization either to standard dosing (control group) or post-transplantation dosing guided by a 2-hour (C2) level following a preoperative tacrolimus dose (T2 group). Methods:  The first postoperative tacrolimus dose was given either according to standard care (control group) or 0.15 mg/kg b.d. if the pre-transplant C2 level was ≤20 ng/mL, 0.1 mg/kg b.d. if the C2 level was 21–59 ng/mL or 0.05 mg/kg b.d. if the C2 level was ≥60 ng/mL (T2 group). Subsequent dosing in both groups was based upon tacrolimus trough level monitoring. Participants received concomitant mycophenolate mofetil and steroids. Results:  Ninety patients were recruited, of which 84 were included in the analysis (control group n = 43; T2 group n = 41). There was no

difference in the proportion of subjects achieving tacrolimus trough levels ≥10 ng/mL (82.9% Control vs Tyrosine-protein kinase BLK 93.0% T2; P = 0.19) or between 10 and 15 ng/mL (41.5% Control vs 41.9% T2; P = 0.97) at day 3 post transplant. The T2 group achieved tacrolimus trough levels of ≥10 ng/mL significantly faster than the control group (100% achievement in 14 days (Control) versus 4 days (T2); P = 0.01). Conclusion:  Performing a pre-transplant tacrolimus C2 does not significantly increase the high proportion of subjects achieving 10 ng/mL tacrolimus concentrations by day 3 using routine protocols. However, compared with standard care, performing a pre-transplant tacrolimus C2 does lead to patients achieving a whole-blood concentration of ≥10 ng/mL sooner.

53–19.41 sec) and 19.81 sec for passages click here (range = 18.31–20.59 sec). The average duration of the Canadian speaker’s stimuli was 17.33 sec for target word lists (range = 16.85–17.80 sec) and 20.24 sec for passages (range = 18.77–21.53 sec). An important consideration is how the speakers used in this work compare with those in the cross-accent experiments of Schmale and Seidl (2009). As noted earlier, the 9-month-olds’ failure to recognize words across a native and a Spanish-accented speaker in Schmale and Seidl may have been owing to the accents varying on several suprasegmental and subphonemic dimensions. In contrast, the speakers used here were predicted to deviate primarily on vowel implementation. Thus,

an examination of acoustic and perceptual differences between these speakers increases

our understanding of the type of variation present in these stimuli, and may shed light on the causes of the 9-month-olds’ failure in previous work. Acoustic measurements and analyses of variance (ANOVAs) with F1 and F2 in /ae/ and /I/ as dependent measures and talker (North Midland-American speaker [“MidW”], and either Spanish-accented speaker (“Span”) or Southern Ontario Canadian speaker [“Can”]) support the prediction that talkers would differ on vowel implementation, see Figure 1, particularly with respect to the backing of /ae/ by the Canadian speaker.2 These dialectal accents Talazoparib nmr were chosen because they should diverge minimally, unlike in nonnative speech, which should diverge at other levels (including general characteristics, such as fluency, and subphonemic characteristics, such as coarticulation). This claim is supported by an investigation of the rate of speech, voicing, and coarticulation of the three speakers, which show that Sitaxentan the MidW and Can speakers differ less than the MidW and Span speakers, as evident in Figure 2. First, nonnative speakers lack

the fluency that characterizes native speakers, which affects global characteristics, including speech rate (although individual variation exists; naturally, a comparison with someone who stutters would not reveal this native advantage). For example, Span exhibited a relatively constant speech rate, whereas the native speakers differ less from each other by talking much slower when uttering words in isolation (I) than within passages (P); ANOVAs with rate as outcome and talker (Midwestern and either Canadian or Spanish) and type (passage, isolation) as factors confirm that the interaction talker × type is much larger in the MidW-Span comparison, F(1, 156) = 32.01 for MidW-Span; 5.34 for MidW-Can. As for consonants, the Spanish-accented speaker produces the /k/ in candle and kingdom with a much shorter VOT than either of the English-speaking speakers, and the VOT differs more, F(1, 78) = 120.72, than in the comparison among the native talkers, F(1, 78) = 27.87.

6). This implies that TAMs in colorectal cancer possess a greater capacity to present antigen and co-stimulate T cells than TAMs in other cancers. To assess the functional capacity of colorectal TAMs in co-stimulating T cells, we performed an MLR assay. TAMs were sorted from colorectal co-culture spheroids and incubated

with allogeneic T cells for 4 days, after which T-cell proliferation was measured by tritiated-thymidine Ku-0059436 purchase incorporation. Indeed, the TAMs were highly competent at stimulating T-cell proliferation (Fig. 4B). Tumour cells sorted from the co-cultures were unable to stimulate T-cell proliferation, indicating that tumour cells per se do not possess T-cell co-stimulatory properties, and in vitro differentiated macrophages were poor stimulators. Together, these observations indicated that TAMs acquired T-cell co-stimulation capabilities during the co-culture with colorectal tumour cells. Of the T cells that proliferated upon incubation

with TAMs, 71% expressed Luminespib in vivo CD25, an activation marker, and 62% produced IFN-γ, a type-1 inflammatory cytokine (Fig. 4C), indicating that TAMs were able to activate type-1 T cells. There was no activation of type-2, type-17 or regulatory-T cells, indicated by the lack of IL-4, IL-17A or FoxP3 (Fig. 4C and D). Together, these results illustrated that TAMs in the colorectal cancer model were capable of stimulating T-cell proliferation and promoting type-1 Isotretinoin T-cell responses. To confirm the in vitro findings on colorectal TAMs, we studied primary tumour tissues from five colorectal cancer patients (Table 1). Pro-inflammatory TAMs were detected in the colorectal tumour sections, as they stained positive for IFN-γ (Fig. 5A, white arrows). The percentage of TAMs that were IFN-γ+ in each tumour sample was quantified using the software TissueQuest, on five images (each ∼350×250 μm) randomly taken from each tumour tissue section. The images

were analysed together to give a representative plot for every tumour sample (Supporting Information Fig. 7). This approach takes into account variations from different parts of the tissue section. The percentage of macrophages that were IFN-γ+ in the tumour samples varied from 6.6 to 50% (Fig. 5B and Table 1). To confirm the in vitro findings that TAMs in colorectal cancers could attract T cells, we quantified the numbers of tumour-infiltrating T cells and TAMs. Indeed, the numbers of tumour-infiltrating T cells and TAMs were highly correlated (r2=0.66, Fig. 5C). Furthermore, the TAMs and T cells were often observed to be in close contact (Fig. 5D, black arrows), suggesting direct interaction of the two cell types, such as antigen presentation to and co-stimulation of T cells by TAMs.

In other words, cholesterol stimulated EPS production. Free bile salts are less soluble than conjugated bile salts, resulting in lower absorption in the intestinal lumen. Deconjugation of bile acids can reduce serum cholesterol levels by increasing the formation of new bile acids that are needed to replace those that have escaped the enterohepatic circulation (30). L. delbrueckii subsp. bulgaricus B3 strain, which has the highest EPS production and cholesterol removal capacity, was selected for the immobilization Pifithrin-�� study. The results may indicate that an interaction occurs between the alginate used for immobilization and the cholesterol in the medium.

In other words, theoretically, cholesterol could be bound to immobilization material. However, to the best of our knowledge, there are no published reports on cholesterol removal by immobilized cells. Results of this study indicate that the use of immobilized probiotic strains, which is effective for cholesterol removal, has a positive influence on cholesterol removal features of the organisms. The results R788 mouse further suggest that immobilized

B3 cells are more resistant to 3 mg/ml oxgall concentration than the free cells. Chandramouli et al. (31) reported that when the immobilized test bacteria were subjected to high bile concentration (1 mg/100 ml bile) there was a significant increase in viable cell counts compared to the free cells under similar conditions. Thus, the immobilization method described in this study may be effectively used to protect the viability and probiotic features of the strains. To the best of our knowledge, the literature

contains no reports on cholesterol removal by Lactobacillus bacteria of 3-oxoacyl-(acyl-carrier-protein) reductase yoghurt origin. The cholesterol removal mechanism by binding or adhering to the bacteria cells, especially to the EPS produced by the bacteria and surrounding the bacterial cells as a capsule, has potential importance in the control of serum cholesterol concentration in humans. In our study, all of the Lactobacillus stains tested removed cholesterol from media during growth. Among them, L. delbrueckii subsp. bulgaricus B3, which has distinctive features in EPS production and cholesterol removal capacity, removed the highest amount of cholesterol. Furthermore, the immobilized B3 strain efficiently reduced cholesterol in the growth medium and, also, the immobilization process raised the bile tolerance of the cells. Results of the present study suggest that immobilized B3 cells have several advantages over the free counterparts. Based on these findings, the combination of a probiotic culture that can remove cholesterol and a strain that has high EPS production capacity could be used to manufacture a fermented dairy product that would have enhanced anti-cholesterolemic activity. Some parts (isolation of cultures and EPS production) of this research were supported by TUBITAK (TBAG-2090(101T129)).

Groups of mice were treated daily for 6 days with fusion protein, treated with vehicle, or untreated as indicated in the legend of Fig. 6. On day 7, the animals were killed; the omenta were removed and treated with collagenase, then stained for flow cytometry as described previously with minor modifications.32 Preliminary RG7204 order experiments were performed using normal omental cells, tumour cells and a reconstructed mixture of tumour cells and omental cells to establish the gates shown. Colony-forming assays were performed as described previously.33 Statistical analyses testing for significance were performed as indicated in the figure legends. We set out to create a cytokine fusion protein that could be cleaved

by a tumour cell expressed protease so that it becomes more active after cleavage. We initially tested a strategy based on steric hindrance learn more by constructing

a fusion protein consisting of IL-2 and Mip1-a separated by a PSA cleavage sequence.34 We hypothesized that both immunomodulatory proteins would be largely inactive in the fusion protein because of their close proximity, but would become more active if the fusion protein could be successfully cleaved, thereby separating the two proteins. Although the fusion protein could be expressed and cleaved by PSA, IL-2 did not appear to be attenuated in the intact fusion protein and the biological activity of the IL-2 did not increase after cleavage (data not shown). Hence, simply joining two molecules, even very closely, does

not necessarily interfere with their functional activity. However, we reasoned that if we constructed a molecule in which the putative inhibitory portion of the fusion protein bound the cytokine specifically, this would be more likely to inhibit its activity. As we describe below, we used two distinct strategies to inhibit the biological activity of IL-2. The first strategy employed a cytokine receptor whereas the second used an antibody fragment (scFv). The first strategy using specific inhibition employed IL-2 and a portion of the IL-2 receptor is illustrated schematically in Fig. 1(a). We used mouse IL-2 cDNA and took advantage of Sulfite dehydrogenase the alpha chain of the IL-2 receptor (IL-2Rα) which can bind IL-2 in the absence of the other subunits (β and γ) of the high-affinity IL-2 receptor.35 In this construct, we eliminated the transmembrane and cytoplasmic region of the IL-2Rα chain, creating a soluble form of the receptor. To increase flexibility and allow the IL-2Rα portion of the molecule to fold back and inhibit IL-2, we also introduced a repeating Gly–Ser linker consisting of (GGGGS)2 (designated 2 ×), or (GGGGS)436 (designated 4 ×), and in some cases also added a 6 × His tag. These plasmids were used to construct recombinant baculoviruses to mediate expression in insect cells as described in the Materials and methods. As shown in Fig. 1(b), we examined the fusion proteins with a capture ELISA using antibodies reactive with IL-2Rα and IL-2. Also, the immunoblot analysis in Fig.

2,25–27 The selection of appropriate, targeted antimicrobial therapy must accommodate the fact that a variety of Candida species ranging from C. albicans to C. parapsilosis have been recovered from cases of CRMD-related Candida endocarditis. Accordingly, current treatment guidelines15 include the use

of an amphotericin B formulation (e.g. liposomal formulation amphotericin B – 3 to 5 mg kg day−1) with or without 5-flucytosine 25 mg kg−1 qid or an echinocandin agent such as micafungin 100 mg day−1 as primary therapy. With regard to the echinocandins, it is noteworthy that two recent publications19,24 Decitabine ic50 describe the use of these agents in the treatment of Candida endocarditis. Alternative step-down therapy can include fluconazole 400–800 mg daily for stable patients with a susceptible organism and negative blood culture results. Treatment is continued for 4–6 weeks after device removal. In summary, CRMD-associated Candida endocarditis is a rare but potentially life-threatening event, the microbiology can include both common and uncommon Candida mTOR inhibitor species and treatment involves both device removal and well-targeted antifungal therapy. “
“Invasive fungal infections (IFI) lead to morbidity and mortality in neutropenic patients and in allogenic stem

cell transplantation. Serum-based fungal detection assays have limitation of specificity or sensitivity. Studies on fungal DNA detection using real-time PCR in childhood leukaemia are lacking. The aim of this study was to develop sensitive and specific diagnostic tools for IFI in paediatric acute leukaemia patients 3-mercaptopyruvate sulfurtransferase using real-time PCR.

Of 100 randomised paediatric acute leukaemia patients receiving antifungal prophylaxis with voriconazole/amphotericin B, single peripheral whole blood sample in EDTA was used for Pan-AC real-time PCR assay (detects nine Candida and six Aspergillus species) in patients who failed prophylaxis due to proven, probable, possible or suspected fungal infections. PCR results were retrospectively correlated with clinical profile. Real-time PCR test was positive in 18/29 (62%) patients who failed prophylaxis. The only patient with proven IFI (mucormycosis), real-time PCR assay was negative. Real-time PCR was positive in 2/4 (50%) patients with possible and 16/24 (66.6%) suspected IFI and 5/10 (50%) patients with pneumonia. By applying method A/B, sensitivity and positive predictive value could not be commented due to unproven Aspergillus or Candida infections; specificity and negative predictive values (NPV) were 41% and 100% respectively; by method C (included episodes of possible IFI as true positive), sensitivity, specificity, PPV and NPV were 50%, 36%, 11% and 81% respectively. In those with suspected IFI, 8/24 (33.

Animal vital statistics are shown in Table 1. An N of 17 sham and 13 PMMTM-exposed animals were used for the intravital preparation, and an N of 11 sham and 8 PMMTM-exposed animals were used for the isolated arteriole preparation (Table 1). All animal procedures were approved by the WVU Institutional Animal Care and Use Committee. Air was sampled at two sites within 1 mile of an active Fulvestrant cost MTM site (Sundial, WV, USA). PM was collected on 35 mm 5 μm pore

size PTFE fiber-backed filters (Whatman, Springfield Mill, UK, Figure 1A) for 2–4 weeks. Air flow rate across the filters averaged 12 L/min. Following collection, the filters were stored at room temperature (20–25°C) and ambient humidity (10–30%) in the dark for 0.5–1 year prior to extraction. PM (Figure 1B) was removed from the filters by gentle agitation in 15 mL of ultrapure water (Cayman Chemical, Ann Arbor, MI, USA) in a glass jar for 96 hours. Storage and extraction of the particles from the filters are consistent with previously reported methods [14]. Aliquots of the particle suspension were dried down in 2 mL cryovials for 18 hours in a Speedvac (Savant, Midland, MI, USA). Total particle weight was determined by a microbalance (Metler-Toledo, Columbus,

OH, USA). Elemental concentration in atomic weight (ppm) was obtained from individual particles with a SEM (JEOL LTD., Tokyo, Japan) coupled with EDX technology (Oxford Instruments, Oxfordshire, UK). A filter sample (˜2 cm2) was obtained from a PTFE filter and mounted with double-sided Phospholipase D1 adhesive copper tape on a brass (Cu and Zn) specimen stub. Approximately four to five samples per Transferase inhibitor filter and 20–25 individual particles per sample were randomly chosen for a total analysis of 100 particles per filter using the Spot & ID EDX Analysis Mode. An accelerating voltage of 20 kV was used and the working distance was set to 15 mm with a 120 seconds live time for X-ray acquisition. Particles 0.5–20 μm were analyzed and a quant optimization was performed on Cu. Analyses were performed on the PMMTM by a commercial laboratory (RTI International,

RTP, NC, USA). Briefly, pre-weighed PMMTM was resuspended into 5 mL of methanol and vortexed. The sample was then split into two equal volume aliquots for ICP-AES and IC analysis. ICP-AES analysis was performed via EPA method 3060C on material extracted using EPA method 3052. Sulfate IC analysis was performed by EPA method 300.0 with modifications for use on the Dionex ICS-3000 (Thermo Scientific, Sunnyvale, CA, USA) with eluent generation [44]. Standard reference material 1648a (St. Louis, MO, US Urban PM; NIST, Gaithersburg, MD, USA) was used as a quality control. The following metals and compounds were determined: Al, Ba, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Si, Sn, Ti, V, Zn, and SO4. Elements not appearing in Table 2 were below detectable limits.